Our long-range goal is to understand the intracellular signaling pathways that are activated by antigen and cytokine receptors on lymphocytes and how they regulate outcomes such as cell growth, differentiation and effector functions. This proposal focuses on the adapter protein Shc and how it may regulate signaling events downstream of the T cell receptor. Adapter protein Shc contains two distinct domains that can interact with tyrosine phosphorylated proteins, and is itself a target tyrosine phosphorylation downstream of the T cell receptor. We have recently generated a conditional knockout mouse using the Cre/ loxP strategy to ablate Shc expression in the T cell lineage. Using Cre expression directed by the lck proximal promoter, we observe that loss of Shc expression in early thymocytes arrests T cell development at the immature CD4-CD8- double negative stage. We have also generated a conditional transgenic mouse (using a novel Cre/ loxP strategy) that express a phosphorylation-defective dominant negative form of Shc. Inducible expression of the ShcFFF also arrests thymic development, essentially at the same stage as the knockout mice. Thus, the expression of Shc and its tyrosine phosphorylation appear to be essential for thymic T cell development. In this proposal, in Aim 1 we will address which of the known downstream signaling pathways downstream of Shc regulate thymic development at the double negative stage. Specifically, we will look at the Role of the Ras/ Erk pathway. We will use a combination of in vivo and in vitro approaches to look at Ras, Raf and Erk activation regulated through Shc. In Aim2, we will examine the significance of individual tyrosines of Shc in determining thymic selection events. We will also take an unbiased approach to look for Shc interaction partners to define known and previously unknown signaling pathways that might affect thymic development. In the third aim, we will examine the role of Shc in positive selection through inducible deletion of the shc locus after pre-TCR stage, but prior to the DP stage. The relevance of Shc mediated Ras/ Erk activation in regulating positive selection will be addressed. Taken together, these studies should provide a better molecular understanding of Shc in vivo, as well as T cell development and function. Since Shc has been linked to growth factor and cytokine signaling in lymphocytes and many other cell lineages, these studies would also have a broader importance for understanding signaling pathways affecting growth and differentiation.